The endogenous opioid-modulating peptide Neuropeptide FF (NPFF, FLFQPQRFamide) activates Neuropeptide FF Receptors 1 and 2 (NPFFR1 and NPFFR2) and exerts its bio-activities. These two NPFF receptors are predominantly expressed in the central nervous system (CNS), with only minimal presence in the peripheral tissues (Koller et al., 2021). Specifically, NPFF and neuropeptide AF were the preferred endogenous ligands for NPFFR2, while the endogenous peptides, RF-amide-related peptide-1 and -3 (RFRP-1 and RFRP-3), had the higher selectivities toward NPFFR1 (Ayachi and Simonin, 2014). The previous studies suggest that NPFF and its receptor NPFFR2 are involved in pain processing (Kaczynska and Wojciechowski, 2021). In contrast, RFRP-3 and its receptor NPFFR1 can modulate the neuroendocrine function (Higo et al., 2021). Furthermore, it has been discovered that NPFFR1 and NPFFR2 regulate food intake and body weight differently, further underscoring their potential involvement in different biological processes (Zhang et al., 2018).

In the past decade, the function of NPFFRs and their endogenous ligands in the CNS has been studied, primarily focusing on their regulation of opioid-induced analgesia and related side effects (Gibula-Tarlowska and Kotlinska, 2020). It has been shown that NPFF displays two different regulatory effects on opioid analgesia, depending on the route of administration. Intracerebroventricular (i.c.v.) injection of NPFF exhibits anti-opioid effects, while intrathecal (i.t.) injection shows pro-opioid activities (Kaczynska and Wojciechowski, 2021). Additionally, results from subcutaneous (s.c.) injection of the nonselective NPFFR antagonists, which could penetrate the blood-brain barrier (BBB), also revealed the anti-opioid face of NPFF on morphine-induced analgesia, tolerance, hyperalgesia, and addiction (Elhabazi et al., 2012). Altogether, these findings suggest that NPFF and its receptors in the CNS are involved in the modulation of opioid-induced analgesia and related side effects. However, the detailed roles of NPFFR1 and NPFFR2 remain unclear.

At the supraspinal levels, initial studies suggested that the anti- and pro-opioid activity of selective NPFFR1 and NPFFR2 agonists was associated with the degree of opioid analgesia but not strictly related to the selectivity towards the two NPFFR subtypes (Quelven et al., 2005). The previous studies showed that intracerebroventricular (i.c.v.) injection of RFRP-3 produced hyperalgesic activity or reduced morphine-induced analgesia, implying the anti-opioid activities mediated by supraspinal NPFFR1 (Elhabazi et al., 2013; Quillet et al., 2021). Furthermore, recent research indicated that the anti-opioid effect of NPFF was mediated through NPFFR2 rather than NPFFR1 (Quillet, et al., 2021). At the spinal level, it is suggested that NPFFR2 was mainly involved in the pharmacological effects of NPFF due to the scarcity of NPFFR1 binding sites in rodent spinal cord. However, this hypothesis has not been definitively supported (Nguyen et al., 2020). Overall, these studies emphasize the significant impact of central NPFF and related peptides system on opioid-induced analgesia and side effects, but the details of which will need further elaboration, especially the specific role of each NPFFR subtype.

Therefore, in this study, pharmacological and genetic inhibition of NPFFR were used to elucidate the modulating roles of central NPFFR1 and NPFFR2 in opioid-induced analgesia and hyperalgesia in mice.